A peptidomics-based investigation of the synergistic effects of neutral and Alcalase proteases on release of antioxidant peptides from soybean protein isolates

Alcalase and neutral protease are widely employed for producing soybean antioxidant peptides, but their synergy mechanisms remain unclear. In this work, soybean protein isolate (SPI) was hydrolyzed synergistically by the two proteases. The products by Alcalase exhibited a larger proportion of low molecular-weight (MW, <1 kDa) peptides compared to those from neutral protease (87.74 % vs 76.08 %). After synergistic hydrolysis, an “averaging” effect on free amino acid composition and MW distribution was observed, but the peptide length distribution shifted evidently towards dipeptides (57.07 % with simultaneous enzyme use, compared to 20.43 % and 33.70 % for individual enzymes). Similarly, the N/C-terminal residue distribution also displayed an ‘averaging’ trend. Furthermore, synergistic processing increased the proportion (approximately 7 %) of potential antioxidant peptides with predicted probability >0.7, aligning with observed non-additive antioxidant activities and suggesting a synergistic mechanism beyond simple additivity. We hope the work could provide guidance and references for the target peptide release from SPI.